Peptidase inhibitors from the salivary glands of the cockroach Nauphoeta cinerea

Inhibitory activity against subtilisin, proteinase K, chymotrypsin and trypsin was detected in the salivary glands and saliva of the cockroach Nauphoeta cinerea (Blattoptera: Blaberidae). Fractionation of the salivary glands extract by affinity chromatography followed by reverse-phase HPLC yielded five subtilisin-inhibiting peptides with molecular masses ranging from 5 to 14 kDa. N-terminal sequences and subsequently full-length cDNAs of inhibitors designated NcPIa and NcPIb were obtained. The NcPIa cDNA contains 216 nucleotides and encodes a pre-peptide of 72 amino-acid residues of which 19 make up the signal peptide. The cDNA of NcPIb consists of 240 nucleotides and yields a putative secretory peptide of 80 amino-acid residues. Mature NcPIa (5906.6 Da, 53 residues) and NcPIb (6713.3 Da, 60 residues) are structurally similar (65.4% amino acid overlap) single-domain Kazal-type peptidase inhibitors. NcPIa with Arg in P1 position and typical Kazal motif VCGSD interacted stoichiometrically (1:1) with subtilisin and was slightly less active against proteinase K. NcPIb with Leu in P1 and modified Kazal motif ICGSD had similar activity on subtilisin and no on proteinase K but was active on chymotrypsin.     

Estimation of population structure in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] using allozyme and microsatellite markers

Characterizing population structure using neutral markers is an important first step in association genetic studies in order to avoid false associations between phenotypes and genotypes that may arise from non-selective demographic factors. Population structure was studied in a wide sample of ∼1,300 coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] trees from Washington and Oregon. This sample is being used for association mapping between cold hardiness and phenology phenotypes and single-nucleotide polymorphisms in adaptive-trait candidate genes. All trees were genotyped for 25 allozyme and six simple sequence repeat (SSR) markers using individual megagametophytes. Population structure analysis was done separately for allozyme and SSR markers, as well as for both datasets combined. The parameter of genetic differentiation (θ or F _ST) was standardized to take into account high within-population variation in the SSR loci and to allow comparison with allozyme loci. Genetic distance between populations was positively and significantly correlated with geographic distance, and weak but significant clinal variation was found for a few alleles. Although the STRUCTURE simulation analysis inferred the same number of populations as used in this study and as based on previous analysis of quantitative adaptive trait variation, clustering among populations was not significant. In general, results indicated weak differentiation among populations for both allozyme and SSR loci (θ _s = 0.006–0.059). The lack of pronounced population subdivision in the studied area should facilitate association mapping in this experimental population, but we recommend taking the STRUCTURE analysis and population assignments for individual trees (Q-matrix) into account in association mapping.     

Superhelical DNA studied by solution scattering and computer models

We present here recent results on the structure of superhelical DNA and its changes with salt concentration between 0.01 and 1.5 M NaCl. Scattering curves of two different superhelical DNAs were determined by static light scattering. The measured radii of gyration do not change significantly with salt concentration. Small-angle neutron scattering, together with calculations from a Monte Carlo model, allows to determine the superhelix diameter. Measured and simulated scattering curves agreed almost quantitatively. Experimentally we find that the diameter decreases from 16.0±0.9 nm at 10 mM to 9.0±0.7 nm at 100 mM NaCl. The superhelix diameter from the simulated conformations decreased from 18.0±1.5 nm at 10 mM to 9.4±1.5 nm at 100 mM NaCl. At higher salt concentrations up to 1.5 M NaCl, the diameter stays constant at 9 nm.This revised version was published online in October 2005 with corrections to the Cover Date.     

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (ΔG° ~ −8.7 to −9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude (ΔG° ~ −1.1 to −1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.     

Large-scale determination of the methylation status of retrotransposons in different tissues using a methylation tags approach

A technique for simultaneous determination of the methylation status of numerous loci containing retroelements (REs) is reported. It is based on the observation that methylated and unmethylated areas in the genome are usually extended, and therefore the methylation of particular methyl-sensitive restriction endonuclease recognition sites might reflect the methylation status of DNA regions around them. The method includes dot-blot hybridization of repeat flanking sequences arrayed on a solid support with specifically amplified flanking regions of presumably unmethylated repeats. A multitude of flanking regions of REs adjacent to unmethylated restriction sites are amplified simultaneously, providing a complex hybridization probe. The technique thus allows the determination of the methylation status of restriction sites, which serve as tags of the methylation status of the surrounding regions. The validity of the technique was confirmed by various means, including bisulfite sequencing. The technique was successfully applied to the identification of methylation patterns of the regions surrounding 38 human-specific HERV-K(HML-2) long terminal repeats in cerebellum- and lymph node-derived genomic DNAs. The described technique can be readily adapted to the use of DNA microarray technology.     

Formation of Palmitic Acid/Ca2+ Complexes in the Mitochondrial Membrane: A Possible Role in the Cyclosporin-Insensitive Permeability Transition

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.     

Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra

It is often assumed mutant frequencies, as measured in a DNA sample, faithfully represent basic mutation rates associated with these mutations. This paradigm was extremely helpful for in vitro studies of the mechanisms of mutagenesis/repair and causes of mutations. However, in vivo, mutant fractions appear to vary dramatically and randomly from sample to sample. It's unlikely that basic mutational rates vary so much. Such variations are probably caused by clonal expansions of mutants within tissue. Whether a particular tissue sample includes an expansion or not, is a matter of chance, which explains the observed random fluctuations of mutant fractions. Well-known examples of clonal expansions involve pathological conditions such as cancer or mitochondrial disease. It is less appreciated that even in normal tissue, expansions of somatic mutants create local deviations from the "expected" mutant frequencies. The sizes of clonal expansions appear to span a wide range and thus, may affect samples of various sizes, from individual cells to individuals. In conclusion, human body appears to be a sort of a "gambling ground" for clonally expanding mutants. We speculate that expansion of early mutants rather than de novo mutation at old age may be the major source of at least some aging-specific mutants in our bodies.